Tumor-associated peptides binding to MHC-molecules

ABSTRACT

The invention relates to a tumor-associated peptide containing an amino sequence, which is selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:79 of the enclosed listing. The peptide has the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I. The invention also relates to the use of the peptides for manufacture of a medicament and for treating tumorous diseases. The invention further relates to a pharmaceutical composition, which comprises at least one of the peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of International Patent ApplicationPCT/EP 03/03181, filed Mar. 27, 2003, designating the United States andpublished in German as WO 03/102023 A1, which claims priority to GermanApplication Number 102 25 144.4, filed Mar. 29, 2002.

TECHNICAL FIELD

The present invention relates to tumor-associated peptides having theability to bind to a molecule of human major histocompatibility (MHC),class I.

Such peptides are used—for example—in immunotherapy of tumor-associateddiseases.

BACKGROUND ART

When tumor cells are eliminated by the immune system the identificationof tumor-associated antigens (TAA) by components of the immune systemplays a pivotable role. This mechanism is based on the fact that thereexist qualitative or quantitative differences between tumor cells andnormal cells. To induce an anti-tumor-response, the tumor cells have toexpress antigens which induce an immune response being sufficient forthe elimination of the tumor.

In particular, CD8-expressing cytotoxic T-lymphocytes (in the followingCTL) are involved in rejection of tumors. To induce such an immunereaction by cytotoxic T-cells foreign proteins/peptides have to bepresented to T-cells. Antigens are recognized as peptide fragments byT-cells only if they are presented by MHC-molecules on cell surfaces.These MHC (“major histocompatibility complex”) molecules are peptidereceptors which normally bind peptides intracellularly and transportthem to the cell surface. This complex of peptide and MHC-molecule isrecognized by T-cells. Human MHC-molecules are also designated as humanleukocyte antigens (HLA).

There are two classes of MHC-molecules: MHC class-I-molecules, which arepresent on most cells having a nucleus, present peptides generated bydegradation of endogenous proteins. MHC class-II-molecules are presenton professional antigen-presenting cells (APC) only and present peptidesof exogenous proteins, which are taken up and processed by APC duringendocytosis. Peptide/MHC-class-I complexes are recognized byCD8-positive cytotoxic T-lymphocytes, peptide/MHC class-II-complexes arerecognized by CD4 helper T-cells.

In order to induce a cellular immune response a peptide has to bind to aMHC-molecule. This action depends on the allele of the MHC-molecule andon the amino acid sequence of the peptide. MHC-class-I-binding peptides,being—as a general rule—of 8 to 10 residues in length, comprise twoconserved residues (“anchor”) in their sequence, that engagecomplementary pockets located in the MHC-molecule.

In order for the immune system to induce an effective CTL-responsedirected against tumor-associated peptides, these peptides have not onlyto be able to bind to specific MHC-class-I-molecules expressed by tumorcells but have also to be able to be recognized by T-cells havingspecific T-cell receptors (TCR).

When developing a tumor vaccine a main aim is to identify and tocharacterize tumor-associated antigens which are recognized by CD8+ CTL.

The antigens—or their epitopes, respectively—which are recognized bytumor-specific cytotoxic T-lymphocytes can be molecules of all classesof proteins, such an enzymes, receptors, transcription factors, etc.Another important class of tumor-associated antigens are tissue-specificstructures such as the cancer-testis antigens, which are expressed invarious kinds of tumors and healthy testis tissue.

In order for the T-lymphocytes to identify proteins as tumor-specificantigens and in order to use them in therapy, certain requirements haveto be met: The antigen has to be expressed mainly by tumor cells and notby normal cells or at least to a minor extent as in tumors. Further, itis desirable if the specific antigen is present not only in one kind oftumor but also in other kinds in high concentrations. Further, thepresence of epitopes in the amino acid sequence of the antigen isessentially since those peptides derived from tumor-associated antigensare supposed to induce a T-cell-response, either in vitro or in vivo.

Thus, TAA represent a starting point for developing a tumor vaccine.Methods for identification and characterization of TAA are based on theutilization of patient-derived CTL or on the generation of differentialtranscription profiles between tumors and normal tissue.

Identification of genes which are overexpressed in tumorous tissues orwhich are selectively expressed in such tissues does not provide preciseinformation about utilization of antigens transcribed from these genesfor immune therapy. This is based on the fact that only several epitopesof these antigens are suitable for such an utilization since a T-cellresponse is induced—via MHC presentation—by epitopes of the antigensonly and not by the antigen as a whole. Thus it is important to selectthose peptides of overexpressed or selectively expressed proteins, thatare presented by MHC-molecules, thereby generating points of attack forspecific tumor recognition by cytotoxic T-lymphocytes.

DISCLOSURE OF THE INVENTION

In view of the above it is an object of the present invention to provideat least one new amino acid sequence of such a peptide which can bind toa molecule of the human major histocompatibility complex (MHC)-class-I.

This object is achieved, according to the invention, by providing atumor-associated peptide containing an amino acid sequence which isselected from the group consisting of SEQ ID NO:1 to SEQ ID NO:79 of theenclosed sequence listing, the peptide having the ability to bind to amolecule of the human major histocompatibility complex (MHC)-class-I.

The object underlying the invention is completely achieved in that way.

It is understood that peptides identified from the tumor may besynthesized or be expressed in cells in order to obtain larger amountsand in order to utilize them for purposes described below.

The inventors were able to identify the above-mentioned peptides asspecific ligands of MHC-class-I-molecules from tumorous tissue. In thisconnection, with the term “tumor-associated”, peptides are denotedherein, which have been isolated and identified from tumorous material.These peptides—being presented on genuine (primary) tumors—are subjectto antigen processing in a tumor cell.

The specific ligands can be used in cancer therapy, for example toinduce an immune response directed against tumor cells, which expressthe corresponding antigens from which the peptides derive.

On the one hand, such an immune response in terms of an induction of CTLcan be achieved in vivo. For that purpose a peptide is administered—forexample in form of a pharmaceutical composition—to the patient, whosuffers from a tumor disease associated with the TAA.

On the other hand, a CTL-response against a tumor expressing the antigenfrom which the peptides derive can be induced ex vivo. For this purposethe CTL-precursor cells are incubated together with antigen-presentingcells and the peptides. The CTL stimulated thereby are then cultivated,and these activated CTL are administered to the patient.

A further possibility is to load APC ex vivo with the peptides and toadminister those loaded APC to a patient, who, in tumor tissue,expresses the antigen from which the peptide is derived. The APC can inturn present the peptide to the CTL in vivo and activate them.

The peptides according to the invention can further be utilized asdiagnostic reagents.

In that way, the peptides can be used to find out if, in a CTLpopulation, there exist CTL specifically directed against the peptide orif the CTL were induced by a therapy.

Further, the increase of precursor T-cells, which show reactivityagainst the defined peptide, can be tested with the peptide.

In addition, the peptide can be used as a marker to assess the diseasecourse of a tumor expressing the antigen from which the peptide derives.

In the enclosed Table 1, the identified peptides are listed. They aredisposed according to the respective HLA-types they are binding to.Further, in the table the proteins are disposed, from which the peptideis deriving, and the respective position of the peptide in thecorresponding protein. In doing so, the English denotation of theproteins was kept to avoid misleading translations. Further, theAcc-numbers are quoted, which are listed in the gene bank of the“National Center for Biotechnology Information” of the NationalInstitute of Health.

The inventors were able to isolate the peptides (or ligands) from renalcell carcinomas of two patients, RCC01 and RCC13. In doing so, 68ligands from tumorous tissue of patient RCC01 were isolated and 13ligands from tumorous tissue of patient RCC13. Two of the ligandsidentified in both patients were identical. Those were the peptideshaving the sequence ID No. 1 and 3 (YVDPVITSI of met-protooncogene(C-Met) and ALLNIKVKL of keratin 18).

79 ligands could be identified from the tumors of the patients, 30 ofwhich were bound to the HLA-subtypes HLA-A*02, 13 were bound toHLA-A*68, 34 to HLA-B*18 or HLA-B*44 and 2 to HLA*24.

HLA-A*02-ligands were all exhibiting the allele-specific peptide motif:(Leucine/Valine, Isoleucine, Alanine or Methionine on position 2;Leucine/Valine, Isoleucine or Alanine at the C-terminus.

Some of the ligands derived from abundantly expressed so-calledhousekeeping genes, which are expressed equally in most tissues, butmany were distinguished by tumor-association.

The peptide having the sequence ID No. 1 YVDPVITSI, for example, isconcerning a ligand, which, in particular, is associated with tumors andwhich derives from the met-protooncogene (c-Met) (position 654-662).Peptides having the sequence ID Nos. 2, 22 and 23 derive fromadipophilin (also denoted as “adipose differentiation related” protein)and comprise positions 129-137, 62-71 and 349-358 in this protein,whereby the last two are among HLA-A*68 presented peptides. The ligandhaving the sequence ID No. 3 is a ligand, which is derived from keratin18 and is located at position 365-373.

The major part of the ligands was comprising the amino acid glutamicacid (E) on position 2, which is an anchor-amino acid of theHLA-B*44-subtype. In that way, peptides could be identified, whichderive from proteins, that have proven to be immunogenic in earlierexperiments, for example peptide having sequence ID No. 5, which derivesfrom protein Annexin II (position in Annexin II: 55-63). This proteinproved to be immunogenic in respect of MHC class-II-molecules inmelanoma patients (see Heinzel et al., The self peptide annexin II(208-223) presented by dendritic cells sentisizes autologous CD4+T-lymphocytes to recognize melanoma cells, 2001, Cancer Immunol.Immunother. 49: 671-678).

Further, some peptides could be identified, which derive from proteins,that are, in particular, overexpressed in tumorous tissue. Thus,fragments of Vimentin (EEIAFLKKL, position 229-237) and Caldesmon(DEAAFLERL, position 92-100) could be identified. Young et al.,Expression profiling of renal epithelial neoplasms: a method for tumorclassification and discovery of diagnostic molecular markers, 2001, Am.J. Pathol., 158: 1639-1651) disclosed that these proteins wereoverexpressed in renal cell carcinoma tissues.

The inventors were further able—among other things—to identify ligands,which derived from ets-1 (NEFSLKGVDF, position 86-95), Alpha-Catenin(NEQDLGIQY, position 169-177) and Galectin 2 (SEVKFTVTF, position80-88).

The inventors further isolated fragment YYMIGEQKF (sequence ID No. 79)which derives from the enzyme Nicotinamid-N-Methyltransferase (position203-211). Takahashi et al., Gene expression profiling of clear cellrenal cell carcinoma: gene identification and prognostic classification,2001, Proc. Natl. Acad. Sci. USA, 98: 9754-9749, disclosed that thisenzyme was overexpressed in renal cells carcinoma.

Surprisingly, the inventors were able to detect cytotoxic T-lymphocytesspecific for one of the identified peptides in donor blood. Thus, it ispossible to induce a CTL-response specific against the tumors.

The inventors were able to demonstrate, in their own experiments, thatby using two exemplarily selected peptides cytotoxic T-lymphocytes (CTL)could be generated in vivo, which were specific for peptides havingsequence ID No. 1 (c-Met-protoocogene-fragment or c-Met-peptide) orspecific for the peptide having the sequence ID No. 2 (adipophilinfragment or adipophiline peptide). These CTL were able to specificallykill tumor cells, which expressed the respective proteins and whichderived from different tumor cell lines of different patients. Theinventors could further demonstrate that with the mentioned CTLdendritic cells, for example, could be lysed, which were previouslypulsed (loaded) with the respective peptides. The inventors demonstratedwith these experiments that human T-cells can be activated in vitro byusing the peptides according to the invention as epitopes. The inventorscould not only demonstrate that CTL, which were obtained from peripheralblood mononuclear cells (PBMNC) of a patient and which were specific fora certain peptide, were able to kill cells of the same kind of tumor ofanother patient. The inventors further demonstrated that even cells ofother kinds of tumors could be lysed with these CTL.

A further object of the invention relates to peptides, which may be usedfor stimulation of an immune response, too, which comprise sequence IDNo. 1 to 79 and in the sequence of which at least one amino acid may bereplaced by another amino acid with similar chemical features.

With respect to the respective MHC-subtypes, these are, for example,anchor amino acids, which may be replaced by amino acids with similarchemical features. For example, in peptides, which are associated withMHC-subtype HLA-A*02, Leucine on position 2 may be replaced withIsoleucine, Valine or with Methionine and vice versa, and Leucine at theC-terminus with Valine, Isoleucine and Alanine, which all comprisenon-polar side chains.

Further, it is possible to use peptides having sequence ID Nos. 1 to 79,which comprise at least one additional amino acid at the N- orC-terminus, or in the sequence of which at least one amino acid may bedeleted.

Further, peptides having sequence ID Nos. 1 79 can be used, whichcomprise at least one amino acid being chemically modified.

The varying amino acid(s) is (are) chosen in that way that the variationdoes not effect the immunogenity of the peptide, that is the peptidestill displays a similar binding affinity to the MHC-molecule and theability to stimulate T-cells.

According to the invention, the peptide can be used for treatment oftumor diseases and/or adenomatous diseases.

Tumor diseases to be treated comprise, for example, renal, breast,pancreas, gastric, testis and/or skin cancer. Listing of tumor diseasesis supposed to be merely illustrative and shall not limit the scope ofusage.

The inventors were able to demonstrate, in their own experiments, thatthe peptides according to the invention are suitable for such use. Thus,it was demonstrated that with specifically generated CTL, which werespecific for certain peptides, tumor cells could be effectively andselectively killed.

To use tumor-associated antigens in a tumor vaccine there are, as ageneral rule, several possible forms of application. Tighe, et al.,1998, Gene vaccination: plasmid DNA is more than just a blueprint,Immunol. Today 19(2): 89-97, demonstrated that the antigen could beadministered either as recombinant protein with suitable adjuvants orcarrier systems or—in plasmid vectors—as cDNA encoding the antigen. Inthe latter cases, to induce an immune response, the antigen has to beprocessed and presented by antigen-presenting cells (APC) in thepatient's body.

Melief, et al., 1996, Peptide-based cancer vaccines, Curr. Opin.Immunol. 8: 651-657, demonstrated a further possibility, i.e., to usesynthetic peptides as vaccine.

A further object of the invention relates to the peptide, which can beused in combination with adjuvants, or on its own.

As an adjuvant, the granulocate-macrophage-colony-stimulating-factor(GM-CSF) can be used, for example.

Further examples for such adjuvants are aluminumhydroxide, emulsions ofmineral oils, such as Freund's adjuvants, saponines or siliconcompounds.

Use of adjuvants is of advantage, since the immune response induced bythe peptide can be boosted and/or the peptide can be stabilized.

Another object of the invention relates to the peptide, which isadministered when bound to an antigen-presenting cell.

This step is advantageously since the peptides can be presented to theimmune system, in particular to cytotoxic T-lymphocytes (CTL). In thatway, CTL can identify and specifically kill the tumor cells. Forexample, dendritic cells, monocytes or B-lymphocytes are suitable asantigen-presenting cells for that purpose.

In doing so, the cells are, for example, loaded with the peptides exvivo. On the other hand, the cells may be transfected with DNA or thecorresponding RNA encoding the peptides in order for the peptides beingexpressed on the cells.

The inventors were able to demonstrate, in their own experiments, thatdendritic cells (DC) could be loaded with specific peptides and thatthese loaded dendritic cells activated peptide-specific CTL. That meansthat the immune system can be stimulated to produce CTL directed againstthe tumors which express the respective peptides.

The antigen-presenting cells carrying the peptide may be used either ina direct manner or may be activated with heat shock protein gp96 prioruse. This heat shock protein induces expression of MHC-class-I-moleculesand of costimulating molecules such as B7, and, in addition, stimulatesproduction of cytokins. In that way the induction of immune responses isenhanced all in all.

Yet another object of the invention relates to peptides, which are usedto label leukocytes, in particular T-lymphocytes.

This use is of advantage, if the peptides are used, in a CTL-population,to detect CTL specifically directed against the peptides.

The peptide can further be used as a marker to assess a therapy courseof a tumor disease.

The peptide can also be used for monitoring therapy in otherimmunizations or therapies. In that way the peptide may not only be usedin a therapeutical way but also in a diagnostic way.

A further object of the invention relates to the peptides, which areused for generating an antibody.

Polyclonal antibodies can be obtained, in a general manner, byimmunization of animals by means of injection of the peptides andsubsequent purification of the immunoglobuline.

Monoclonal antibodies can be generated according to standardizedprotocols, for example as described in Methods Enzymol. (1986), 121,Hybridoma technology and monoclonal antibodies.

A further object of the invention relates to a pharmaceuticalcomposition comprising one or more peptides.

This composition may for example be applied parenterally, for examplesubcutaneously, intradermally or intramuscularly or may be administeredorally. In doing so the peptides are dissolved or suspended in apharmaceutically acceptable carrier, preferably an aqueous carrier. Thecomposition can further comprise additives, for example, buffers,binders, diluents etc.

The peptides can also be administered together with immunostimulatingsubstances, for example cytokins. An extensive description of additiveswhich can be used in a composition of this nature is given, for example,in A. Kibbe, Handbook of Pharmaceutical Excipients, 3. ed., 2000,American Pharmaceutical Association and pharmaceutical press.

The composition may be used for prevention, prophylaxis and/or therapyof tumor diseases and/or adenomatous diseases.

The pharmaceutical composition comprising at least one of the peptideshaving sequence ID Nos. 1 to 79 is administered to a patient who suffersfrom a tumor disease, the respective peptide or antigen is associatedwith. Thereby, a tumor-specific immune response can be induced on basisof tumor-specific CTL.

The amount of the peptide or of the peptides being present in thepharmaceutical composition is a therapeutically effective amount. Inthis connection the peptides contained in the composition can bind to atleast two different HLA-types.

The present invention relates, as a further object of the invention tonucleic acid molecules encoding the peptides with sequence ID Nos. 1 to79.

The nucleic acid molecules can represent DNA- or RNA-molecules and canbe used for immune therapy of cancer as well. Thereby the peptideexpressed by the nucleic acid molecule induces an immune responseagainst tumor cells, which express the peptide.

According to the invention the nucleic acid molecules can be provided ina vector.

The invention further relates to a cell genetically modified by means ofthe nucleic acid molecule so that the cell is producing a peptide havingsequence ID Nos. 1 to 79.

For this purpose, the cells are transfected with DNA or correspondingRNA encoding the peptides, thereby expressing the peptides on the cells.For this purpose, for example dendritic cells, monocytes orB-lymphocytes are suitable as antigen-presenting cells.

It will be understood that the features which are mentioned above andthe features still to be explained below can be used not only in thecombinations which are in each case specified but also in othercombinations or on their own without departing from the scope of thepresent invention.

Embodiments of the invention are displayed and explained in the figuresbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the detection of CD8+-T-lymphocytes specific for keratin18;

FIGS. 2 a-d show the induction of CTL-responses in vitro, specific forthe c-Met-peptide (SEQ ID No. 1), FIGS. 2 a and 2 b, or theadipophilin-peptide (SEQ ID No. 2), FIGS. 2 c and 2 d;

FIGS. 3 a-f show antigen-specific lysis of tumor cell lines expressingc-Met or adipophilin, mediated by c-Met-peptide (SEQ ID No. 1), FIGS. 3a-d, or adipophilin-peptide (SEQ ID No. 2), FIGS. 3 e and 3 f, inducedCTL;

FIGS. 4 a-c show lysis-inhibition assays with 51 Cr-labeled tumor cellsand unlabeled pulsed T2-cells mediated by c-Met-peptide (SEQ ID No. 1),FIGS. 4 a and 4 b, or adipophilin-peptide (SEQ ID No. 2), FIG. 4 c)induced CTL;

FIGS. 5 a and b show lysis of autologous dendritic cell transfected withtumor RNA mediated by c-Met-peptide (SEQ ID No. 1), FIG. 5 a, oradipophilin-peptide (SEQ ID No. 2), FIG. 5 b, induced CTL;

FIG. 6 shows that adipophilin-specific autologous CTL induced in vitrorecognize autologous tumor cells of a patient with chronic lymphaticleukaemia but not autologous dendritic or B-cells.

MODES OF CARRYING OUT THE INVENTION EXAMPLE 1

1.1 Patient Samples

Samples of patients having histologically confirmed renal cell carcinomawere obtained from the department of urology, University of Tübingen.Both patients had not received preoperative therapy. Patient No. 1 (inthe following designated RCC01) had the following HLA-typing: HLA-A*02A*68 B*18 B*44; patient No. 2 (in the following designated RCC13)HLA-A*02 A*24 B*07 B*40.

1.2 Isolation of MHC-Class-I-Bound Peptides

Shock-frozen tumor samples were processed as described in Schirle, M. etal., Identification of tumor-associated MHC-class I ligands by a novel Tcell-independent approach, 2000, European Journal of Immunology, 30:2216-2225. Peptides were isolated according to standard protocols usingmonoclonal antibody W6/32 being specific for HLA class I or monoclonalantibody BB7.2 being specific for HLA-A2. Production and utilization ofthese antibodies is described by Barnstable, C. J. et al., Production ofmonoclonal antibodies to group A erythrocytes, HLA and other human cellsurface antigens—New tools for genetic analysis, 1978, Cell, 14:9-20 andParham, P. & Brodsky, F. M., Partial purification and some properties ofBB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and avariant of HLA-A28, 1981, Hum. Immunol., 3: 277-299.

1.3 Mass Spectrometry

Peptides from tumor tissue of patient RCC01 were separated by reversedphase HPLC (SMART-system, μRPC C2/C18 SC 2.1/19, Amersham PharmaciaBiotech) and fractions were analyzed by nanoESI MS. In doing so it wasproceeded as described in Schirle, M. et al., Identification oftumor-associated MHC class I ligands by a novel T cell-independentapproach, 2000, European Journal of Immunology, 30: 2216-2225.

Peptides from tumor tissue of patient RCC13 were identified by onlinecapillary LC-MS as mentioned above with minor modifications: Samplevolumes of about 100 μl were loaded, desalted and preconcentrated on a300 μm*5 mm C18 μ-precolumn (LC packings). A syringe pump (PHD 2000,Harvard Apparatus, Inc.) equipped with a gastight 100 μl-syringe (1710RNR, Hamilton), delivered solvent and sample at 2 μl/min. For peptideseparation, the preconcentration column was switched in line with a 75μm*250 mm C-18-column (LC packings). Subsequently a binary gradient of25%-60% B within 70 min was performed, applying a 12 μl/min flow ratereduced to approximately 300 nl/min with a precolumn using a TEE-piece(ZT

IC, Valco) and a 300 μm*150 mm C-18-column.

A blank run was always included to ensure that the system was free ofresidual peptides. On-line fragmentation was performed as described andfragment spectra were analyzed manually. Database searches (NCBInr, EST)were made using MASCOT.

1.4 Identification of 77 MHC-Class-I-Ligands of Yumorous Tissue ofPatient RCC01

In the enclosed Table 1 the ligands are listed which were bound toHLA-A*02, HLA-A*68, HLA-B*18 or HLA-B*44. Peptides that bound toHLA-A*02 reflected the allele-specific peptide motif: On position 2Leucine, Valine, Isoleucine, Alanine or Methionine and at the C-terminusLeucine, Valine, Isoleucine or Alanine. Most ligands were derived fromso-called housekeeping proteins but ligands from proteins with reportedtumor-associated associations could be detected also.

HLA-A*68 ligands were identified by their anchor amino acid Threonine,Isoleucine, Valine, Alanine or Leucine on position 2 and Arginine orLysine at the C-terminus. This indicated to subtype HLA-A*6801. Twoother ligands from adipophilin were found among HLA-A*68 presentedpeptide, MTSALPEIQK and MAGDIYSVFR, and further ETIPLTAEKL deriving fromtumor-associated cycline D1. Peptide TIVNILTNR derives from Annexin II,this protein proved as immunogenic in connection with MHC-class-II inmelanoma patients (see Heinzel et al., The self peptide annexin II(208-223) presented by dendritic cells sentisizes autologous CD4+T-lymphocytes to recognize melanoma cells, 2001, Cancer Immunol.Immunother. 49: 671-678). Further ligands were carrying glutamic acid onposition 2 which is an anchor amino acid of the HLA-B*44-subtype. Sincethe peptide motifofHLA-B*18 is unknown the distinction between ligandsof these two HLA-B-molecules was not possible.

1.5 MHC-Class-I-Ligands of Tumorous Tissue of Patient RCC13

With this tumorous tissue, too, the same ligands could be identified,which have been identified in patient RCC01 and which derived frommet-protooncogene (c-Met) and keratin 18: peptides having sequence IDNos. 1 and 3. In addition, further ligands could be obtained from thistumorous tissue: A ligand could be identified which derives fromnicotinamide-N-methyltransferase (NNMT); this gene is overexpressed inmore than 95% of all renal carcinoma. Further, some other ligandsoverlap with the peptide repertoire of RCC01.

1.6 Detection of Keratin 18-Specific T-cells in Normal CD8⁺-T-CellRepertoire

Peripheral blood mononuclear cells from healthy patients were stainedwith HLA-A*0201-tetramers which were folded with adipophilin-, keratin18- or met-protooncogene (c-Met)-peptides: For generation of thetetramers, recombinant HLA-A*0201-molecules were folded in vitro withthe peptides SVASTITGV (SEQ ID No. 2, adipophilin), ALLNIKVKL (SEQ IDNo. 3, keratin 18) or YVDPVITSI (SEQ ID No. 1, met-protooncogene,c-Met), purified by means of gel filtration, biotinylated and mixed withstreptavidin to link the monomers.

Unexpectedly, a significant population of CD8⁺-T-lymphocytes specificfor keratin 18 was found in four out of 22 healthy individuals. In FIG.1, the results of double staining are shown in dotplots, whereby in themiddle row the results of staining with keratin 18 is shown. Between0.02 and 0.2% of the CD8⁺-positive T-cells were specific for keratin 18.As can be seen from the lower row of the dotplots, binding of thekeratin 18-tetramer was specific.

EXAMPLE 2

To analyze presentation of the peptides with SEQ ID No.1 (YVDPVITSI)(peptide fragment of c-Met-protooncogene) and SEQ ID No. 2 (peptidefragment of adipophilin) by tumor cells and their recognition by CTL,CTL were induced in vitro, which were specific for the c-Met-peptide(peptide with SEQ ID No. 1) and CTL which were specific for theadipophilin-peptide (SEQ ID No. 2). In doing so, dendritic cells (DC)derived from healthy HLA-A*02-positive donors were used.

2.1 Generation of DC

Peripheral blood mononuclear cells (PBMNC) were isolated byFicoll/Paque-(Biochrom, Berlin, Germany)-density gradientscentrifugation of heparinized blood obtained from buffy coatpreparations of healthy volunteers from the blood bank of the Universityof Tübingen. Cells were seeded (1×10⁷ cells/3 ml per well) into 6-wellplates (Falcon, Heidelberg, Germany) in RP10 media (RPMI 1640, suppliedwith 10% heat-inactivated fetal calf serum and with antibiotics). After2 hours of incubation at 37° C. and 5% CO₂, non-adherent cells wereremoved and the adherent blood monocytes were cultured in RP10 mediumsupplemented with the following cytokins: human recombinant GM-CSF(granulocyte makrophage colony stimulating factor; Leukomax, Novartis;100 ng/ml), Interleukin IL-4 (R&D Systems, Wiesbaden, Germany, 1000IU/ml) and TNF-α (tumor necrosis factor α) (R&D Systems, Wiesbaden,Germany, 10 ng/ml).

2.2 Synthesis of Peptides

Exemplary, two HLA-A*02-binding peptides (c-Met SEQ ID No. 1, YVDPVITSI)or adipophilin (SEQ ID No. 2, SVASTITGV) which were identified asdescribed above) were synthesized using standard F-moc chemistry on apeptide synthesizer (432A, Applied Biosystems, Weiterstadt, Germany) andanalyzed by reversed phase HPLC and mass spectrometry. In that way,sufficient amounts of the identified peptides can be generated.

2.3 Induction of Antigen-Specific CTL-Response using HLA-A*02 RestrictedSynthetic Peptides

For CTL induction, the DC obtained in step 2.1 (5×10⁵) were pulsed withthe peptides obtained in step 2.2 with SEQ ID No. 1 or SEQ ID No. 2,each with 50 μg/ml for 2 hours, washed and incubated with 2.5×10⁶autologous PBMNC in RP10 medium.

After 7 days of culture, cells were restimulated with autologous PBMNCpulsed with peptides. In doing so, 1 ng/ml human recombinant InterleukinIL-2 (R&D Systems) was added on days 1, 3 and 5. The cytolytic activityof thereby induced CTL was analyzed on day 5 after the lastrestimulation in a standard ⁵¹Cr-release-assay (see below, under 2.4:CTL-assay).

2.4 CTL-Assay

In the CTL-assays, tumor cells, peptide-pulsed cells of different celllines and autologous DC were used as target cells. Peptide-pulsed cellswere pulsed with 50 μg/ml peptide (SEQ ID No. 1 or SEQ ID No. 2) for 2hours. All target cells were labeled with [⁵¹Cr] sodium chromate in RP10(RPMI 1640, supplemented with 10% heat inactivated calf serum andantibiotics) for 1 hour at 37° C. Subsequently, 10⁴ cells/well weretransferred to a 96-well round bottomed plate. Varying numbers of CTLwere added to give a final volume of 200 μl and incubated for 4 hours at37° C. At the end of the assays, supernatants (50 μl/well) wereharvested and counted in a beta-plate counter. The percent-specificlysis was calculated as: 100×(experimental release−spontaneousrelease/maximal release−spontaneous release). The spontaneous andmaximal release were determined in the presence of either medium or 2%Triton X-100, respectively.

2.5 Results of the CTL-Induction

a) CTL-cytotoxic Activity Against Peptide-Pulsed DC

In FIG. 2, the results of the ⁵¹Cr-release-assay (see under 2.4) withrespect to the cytotoxic activity of induced CTL (see under 2.3) againstT2- or DC-cells is shown. The T2-cell line is HLA-A*02-positive and TAP(transporter associated with antigen processing) deficient;(TAP-peptide-transporter transport peptide fragments of a proteinousantigen from the cytosol to the endoplasmatic reticulum, where theyassociate with MHC-molecules).

In FIGS. 2 a and 2 b, the cytotoxic activity of CTL induced with peptidewith SEQ ID NO: 1 against T2-cells and DC is shown, both cell types hadpreviously been pulsed with the (c-Met-)peptide with SEQ ID No. 1 (blackfilled boxes) or an ir-relevant peptide (Survivin(=“Sv”; ELTLGEFLKL; SEQID No. 80) or HIV (ILKEPVHGV; Pol. HIV-1 reverse transcriptase peptide,position 476-484; SEQ ID No. 81). In FIGS. 2 c and 2 d, the cytotoxicactivity of CTL induced with peptide with SEQ ID NO:2 against T2- andDC-cells is shown, which had previously been pulsed with the(adipophilin)-peptide with the SEQ ID No. 2.

The specific lysis, which is demonstrated in the release of ⁵¹Cr, is, inFIGS. 2 a-2 d,—as well as in the CTL-lysis-diagrams of FIGS. 3-5—shownvs. different ratios of effector cells (CTL) to target cells(⁵¹Cr-labeled cells to be lysed).

As can be seen from FIGS. 2 a -2 d, an antigen-specific killing of cellscould be demonstrated with a CTL-cell line, which has been generatedafter 2-weekly restimulation: Only cells were lysed by an increasingamount of CTL, which presented either the c-Met-peptide with the SEQ IDNo. 1 (FIGS. 2 a and 2 b) or the adipophilin-peptide with the SEQ IDNO:2 (FIGS. 2 c and 2 d) (see in the FIGS. 2 a -2 d curves with blackfilled boxes, respectively); while control cells pulsed with irrelevantpeptides were not lysed (curves with empty boxes). Thereby thespecificity of the cytolytic activity could be demonstrated.

b) CTL-cytotoxic Activity Against Tumor Cell Lines

Next, it was tested, in a standard tumor ⁵¹Cr-release-assays again,whether CTL specific for the c-Met-peptide with SEQ ID No. 1 or foradipophilin-peptide with SEQ ID No. 2 recognized and lysed tumor cells,which endogeously express the c-Met-protooncogene or adipophilin.

In doing so, the following cell lines, ⁵¹Cr-labeled, HLA-A*02-positive,were used: HCT 116 (colon cancer; obtained from Prof. G. Pawelec,Tübingen, Germany), A 498, MZ 1257 and MZ 1774 (renal cell carcinoma;obtained fro Prof. A. Knuth, Frankfurt, Germany), MCF-7 (breast cancer;obtained from ATCC, American Type Culture Collection), Mel 1479(Malignant melanoma; obtained from Prof. G. Pawelec, Tübingen, Germany)and U 266 (multiple myeloma; obtained from Prof. G. Pawelec, Tübingen,Germany). These cell lines express c-Met-protooncogene and adipophilinas target structures (“targets”).

In the experiments CEBV (Epstein-Barr-virus)-immortalized B-cell lineCroft, HLA-A*01-positive; obtained from O. J. Finn, Pittsburgh, USA) andcell line SK-OV-3 (ovarian cancer; HLA-A*03-positive; obtained from O.J. Finn, Pittsburgh, USA) were used as negative controls. K 562-cells(for example obtainable at the German Collection of Mikro Organisms andCell Cultures DSMZ; ACC 10) were used to determine the activity ofnatural killer cells (NK) since the cell line is highly sensitive forthese killer cells.

All cell lines were cultivated in RP10 medium (RPMI 1640, supplementedwith 10% heat-inactivated fetal calf serum and antibiotics).

With the above-mentioned tumor cell lines and the CTL induced⁵¹Cr-release assays (see under 2.4.) were carried out as mentionedabove.

FIGS. 3 a-3 f show the results of these CTL-assays, whereby in FIGS. 3a-3 d CTL were used which were induced using c-Met-peptide with SEQ IDNo. 1, and in FIGS. 3 e-3 f CTL were used, which were induced usingadipophilin peptide with SEQ ID No. 2.

As can be seen from FIGS. 3 a-3 f, the CTL specific for c-Met-peptidewith SEQ ID No. 1 (FIG. 3 a-3 d) or specific for adipophilin peptidewith SEQ ID No. 2 (FIGS. 3 e and 3 f) were able to efficiently lysetumor cells expressing both HLA-A*02 and c-Met or adipophilin (that isin FIG. 3 a cell line HCT 116, in FIG. 3 b cell line A 498, in FIG. 3 ccell lines MZ 1257 and MEL 1479 and in FIG. 3 d cell lines MCF-7 and U266; in FIG. 3 e cell lines A 494, U 266 and MCF-7, in FIG. 3 f celllines MZ 1774, Mel 1479 and MZ 1257). Specific lysis was measured—asmentioned under 2.4.—via ⁵¹Cr release. There was no lysis of the controlcell line SK-OV-3 (HLA-A-*02-negative), neither through CTL, which wereinduced by the peptide with SEQ ID NO:1 nor through CTL, which wereinduced by the peptide with SEQ ID NO:2. Thus, it could be demonstratedthat both peptides have to be presented on tumor cells in connectionwith HLA-A*02-molecules to efficiently lyse the target cells. Further,the antigen-specificity and the MHC-restriction of the CTL is proved inthat way.

CTL-cells induced in vitro with the peptide having SEQ ID NO:1 did notrecognize the K562 cell line (see FIGS. 3 a, 3 b and 3 d), indicatingthat the cytotoxic activity was not mediated by natural killer(NK)-cells.

c) Inhibition-Assays

To further verify the antigen-specificity and the MHC-restriction of thein-vitro-induced CTL, inhibition assays with non-⁵¹Cr-labeled (“cold”)inhibitor cell lines were performed.

In doing so, the ability of peptide-pulsed cell lines was analyzed toinhibit the lysis of tumor cells (competition assay). For this purpose,an excess of inhibitor (that is an excess of unlabeled pulsed cells) wasused. The ratio of inhibitor (peptide-pulsed cells) to target (tumorcells) was 20:1. When inhibitor cell lines were lysed, no ⁵¹Cr wasreleased, since the inhibitor cell lines were unlabeled.

Cell line T2 (HLA-A*02; TAP-deficient; see under 2.5.a) was used asinhibitor. Previous to the assay, this cell line T2 was pulsed with therelevant peptides (SEQ ID NOS:1 or 2) or an irrelevant control peptide(Survivin(=Sv), SEQ ID NO:80), respectively.

Results of these tests are shown in FIGS. 4 a -4 c, whereby in FIGS. 4 aand 4 b CTL were used which were c-Met-peptide-induced (SEQ ID NO:1) andin FIG. 4 c CTL were used, which were adipophilin-peptide-induced (SEQID NO:2).

In FIGS. 4 a and 4 b, lysis of the ⁵¹Cr-labeled cell lines U 266 and A498 was tested without inhibitor cell line (see the curve with blackfilled boxes); lysis with inhibitor cell line T2, pulsed with anirrelevant peptide (Survivin; SEQ ID NO:80; negative control, curveswith the filled triangles); and lyses with the inhibitor cell line T2pulsed with the c-Met-peptide with SEQ ID NO:1 (curves with the emptyrhombus).

Without inhibitor cells, lysis of tumor cells by CTL could bedemonstrated (see in FIGS. 4 a-4 d curves with black filled boxes,respectively). Further, as can be seen from FIGS. 4 a and 4 b, whenusing an excess of inhibitor target tumor cells were not lysed (and no⁵¹Cr was released), if the inhibitor target was pulsed withc-Met-peptide with SEQ ID NO:1 (see curves with the empty rhombussymbols, respectively). The activity of the CTL was directed against theexcess unlabeled T-cells, so that these cells and not the tumor cellswere lysed. The T2-cells pulsed with an irrelevant peptide (Survivinrespectively; SEQ ID NO:80) were not able to inhibit the lysis of tumorcells by CTL, so that released ⁵¹Cr could be measured (see in FIGS. 4 aand 4 b curves with black filled triangles).

A similar event could be shown when using CTL induced with adipophilinpeptide with SEQ ID NO:2 (see FIG. 4 c):

MHC-restriction and antigen-specificity of the cytotoxic activity of theadipophiline-induced CTL could be demonstrated by using aHLA-A*02-specific monoclonal antibody and in an inhibition assay withunlabeled (“cold”) inhibitor: The results of this experiment are shownin FIG. 4 c. A 498-tumor cells were blocked when addingHLA-A*02-specific antibody (monoclonal antibody BB7.2, IgG2b, obtainedfrom S. Stefanovic, Tübingen) so that they were not lysed by the addedCTL and no ⁵¹Cr was released (see FIG. 4 c curve with filledtriangle-symbols). As a control a non-specific antibody was used, whichdid not block the HLA-A*02-molecule (ChromPure Maus IgG, Dianova,Germany; see in FIG. 4 c curve with filled boxes). With these inhibitionassays, the cells were incubated with 10 μg/ml antibody previous toseeding on the 96-well-plates for 30 min.

Further, it could be demonstrated that the T2-competition cell linepulsed with the irrelevant peptide Survivin (SEQ ID NO:80) (T2/SV), wasnot able to inhibit CTL-induced lysis of tumor cell line A 498 (see inFIG. 4 c curve with black filled circles), but T2-inhibitor cell linepulsed with adipophilin peptide with SEQ ID NO:2 (T2/AD) was able toinhibit the lysis of the tumor cell line, so that—refrain to the lattercase—no ⁵¹Cr release could be measured (see in FIG. 4 c curve withx-symbols).

d) Specific Lysis of Transfected DC

In a next experiment, the cytotoxic activity of CTL in an autologoussetting was analyzed. In doing so, autologous DC, generated from thesame PBMNC that were utilized for CTL induction (see under 2.2.), wereused as target cells. Prior to the CTL-assay, the DC were electroporatedwith RNA, which was previously isolated either from tumor cell lines orwhich represented control-RNA (in vitro transcribed EGFP-RNA, enhancedGreen fluorescent protein-RNA); plasmide: pSP64 Poly(A) EGFPII, obtainedfrom Van Tendeloo, Antwerp, Belgium). Total RNA of tumor cells wasisolated with the QIAGEN Rneasy Mini Kit (QIAGEN, Hilden, Germany)according to the manufacturer's protocol. Quantity and purity of RNAwere determined by spectrophotometry and stored in aliquots at −80° C.

Prior to electroporation on day 6, immature DC were washed twice withserum-free X-VIVO 20 medium (BioWhittaker, Walkersville, USA) andresuspended to a final concentration of 2×10⁷ cells/ml. Subsequently,200 μl of the cell suspension were mixed with 10 μg of total RNA andelectroporated in a 4 mm-cuvette using an Easyject Plus™ (Peglab,Erlangen, Germany) (parameters: 300 V, 150 μF, 1540 Ω, pulse time 231ms). After electroporation the cells were immediately transferred intoRP10 medium and returned to the incubator. More than 80% of the cellsproved to be viable after electroporation.

The results of these experiments are shown in FIGS. 5 a and 5 b. In FIG.5 a CTL were used, which were induced with c-Met-peptide with SEQ IDNO:1, in FIG. 5 b CTL were used which were induced with adipophilinpeptide with SEQ ID NO:2.

After performing the CTL-assay with the CTL induced by c-Met-peptide(SEQ ID NO:1) (see under 2.4.) a specific lysis of DC could bedemonstrated which were electroporated with RNA of c-Met-expressingtumor cell lines (A 498 and MCF-7) (see in FIG. 5 a, curves with blackfilled symbols). DC electroporated with RNA of the non-C-met-expressingtumor cell line Croft were not lysed (see curve with the empty rhombus).

CTL induced with adipophilin-peptide with SEQ ID NO:2 lysed DC whichwere electroporated with RNA of the adipophilin-expressing cell line A498 (see in FIG. 5 b curve with black filled triangles). Further, DCwere lysed which were pulsed with the adipophilin-peptide SEQ ID NO:2(see in FIG. 5 b curve with black filled rhombus). On the other hand, DCelectroporated with control (EGFP) RNA were not lysed (see in FIG. 5 bcurve with the empty triangles).

Thus it could be demonstrated that—after transfection of the DC with RNAof c-Met- or adipophilin-positive tumor cells—the identified peptides,that is c-Met-peptide with SEQ ID NO:1 and adipophilin-peptide with SEQID NO:2, were processed and presented.

e) Induction of Adipophilin-Specific CTL in a Patient with ChronicLymphatic Leukemia

In a further experiment, CTL were generated from PBMNC ofHLA-A*0201-positive patient with chronic lymphatic leukemia (CLL), whichwere specific for adipophilin-peptide with SEQ ID NO:2. The patient wasin remission after treatment with fludarabine. Further, autologousCLL-cells and DC of this patient were used as ⁵¹Cr-labeled targets in anassay, in which ⁵¹Cr-release is mediated by the peptide-induced CTL.

As shown in FIG. 6, the peptide-induced CTL efficiently lysed autologousDC from this patient that were pulsed with the adipophilin peptide withSEQ ID NO:2 (“DC+AD”) as well as the autologous CLL-cells (“CLL cells”).DC which were pulsed with the irrelevant peptide Survivin with SEQ IDNO:80 were—on the other hand—not lysed (“DC+SV”). Also, non-malignantB-cells and cell line K 562 were not lysed by CTL.

The specificity of the CTL-response was further confirmed in a targetinhibition assay, using the cell line T2 (see above) as inhibitor cells,which were pulsed with the adipophilin peptide with SEQ ID NO:2 or withthe irrelevant peptide Survivin with SEQ ID NO:80, respectively. The CTLinduced with adipophilin-peptide with SEQ ID NO:2 lysed the excessinhibitor cell lines which were pulsed with the relevant peptide withSEQ ID NO:2 so that ⁵¹Cr-labeled tumor cells were not lysed in this case(see in FIG. 6 the curve with empty boxes).

In conclusion, the inventors could show that the identified peptidesrepresent promising substances in the scope of an immune therapy formany (tumor) diseases. TABLE 1 Sequence Position/Gene Acc. No. SEQID-No. Patient RCCO1 HLA-A*02 1. YVDPVITSI 654-662 J02958 SEQ ID-Nr. 1met proto-oncogen 2. SVASTITGV 129-137 X97324 SEQ ID-Nr. 2 adiposedifferentiation-related protein 3. ALLNIKVKL 365-373 M26326 SEQ ID-Nr. 3keratin 18 4. ALFDGDPHL 1-9 AB002365 SEQ ID-Nr. 4 KIAA0367 5. RLLDYVVNI679-687 AB040951 SEQ ID-Nr. 5 hypothetical protein FLJ20004 6. ALANGIEEV101-109 AY014906 SEQ ID-Nr. 6 apolipoprotein L, 3 7. QLIDKVWQL 593-601D67029 SEQ ID-Nr. 7 SEC14 (S. cerevisiae)-like 1 8. ALSDLEITL 389-397Z24725 SEQ ID-Nr. 8 mitogen inducible 2 9. ILDTGTIQL 174-182 AB013094SEQ ID-Nr. 9 kidney- and liver-specific gene 10. SLLGGDVVSV 27-36AF153603 SEQ ID-Nr. 10 delta sleep inducing peptide, immunoreactor 11.FLDGNELTL 167-175 U93205 SEQ ID-Nr. 11 chloride intracellular channel 112. NLLPKLHIV 179-187 U93205 SEQ ID-Nr. 12 chloride intracellularchannel 1 13. ALASHLIEA 507-515 AF181263 SEQ ID-Nr. 13 EH-domaincontaining 2 14. SLYGGTITI 296-304 AK000697 SEQ ID-Nr. 14 hypotheticalprotein FLJ11189 15. FLLDKKIGV 218-226 AF026166 SEQ ID-Nr. 15 chaperonincontaining TCP1, subunit 2 (beta) 16. FLDGNEMTL 178-186 AF097330 SEQID-Nr. 16 chloride intracellular channel 4 17. AIVDKVPSV 147-155AF100756 SEQ ID-Nr. 17 coat-protein gamma-cop 18. DVASVIVTKL 241-250U51920 SEQ ID-Nr. 18 signal recognition particle 54kD 19. LASVSTVL130-137 AF230076 SEQ ID-Nr. 19 hemoglobin, alpha 2 20. VMAPRTLVL 3-11SEQ ID-Nr. 20 HLA-A 21. LLFDRPMHV 267-275 L03532 SEQ ID-Nr. 21 hnRNP MHLA-A*68 22. MTSALPIIQK 62-71 X97324 SEQ ID-Nr. 22 adiposedifferentiation-related protein 23. MAGDIYSVFR 349-358 X97324 SEQ ID-Nr.23 adipose differentiation-related protein 24. ETIPLTAEKL 115-124 X59798SEQ ID-Nr. 24 cyclin D1/PRAD1 25. DVMVGPFKLR 934-943 AJ303079 SEQ ID-Nr.25 Akinase (PRKA) anchor protein 2 26. TIIDILTKR 64-72 X05908 SEQ ID-Nr.26 annexin A1 27. TIVNILTNR 55-63 BC001388 SEQ ID-Nr. 27 annexin A2 28.TIIDIITHR 385-393 J03578 SEQ ID-Nr. 28 annexin A6 29. SIFDGRVVAK 107-116AB020980 SEQ ID-Nr. 29 putative membrane protein 30. STIEYVIQR 115-123BC005032 SEQ ID-Nr. 30 Sec23 (S. cerevisiae) homolog B 31. ELIKPPTILR132-141 AF092092 SEQ ID-Nr. 31 adaptor-related protein complex 3 32.EIAMATVTALR 248-258 X12447 SEQ ID-Nr. 32 aldolase A,fructose-biphosphate 33. ETIGEILKK 95-103 BC000355 SEQ ID-Nr. 33 hnRNPK34. SLADIMAKR 86-94 BC000690 SEQ ID-Nr. 34 ribosomal protein L24HLA-B*44 oder HLA-B*18 35. EEIAFLKKL 229-237 M14144 SEQ ID-Nr. 35vimentin 36. DEAAFLERL 92-100 M64110 SEQ ID-Nr. 36 caldesmon 1 37.DEMKVLVL 545-552 M96803 SEQ ID-Nr. 37 spectrin, beta, non-erythrocytic 138. DEVKFLTV 191-198 M82809 SEQ ID-Nr. 38 annexin A4 39. NENSLFKSL935-943 D21260 SEQ ID-Nr. 39 clathrin, heavy polypeptide (Hc) 40.DEFKVVVV 373-380 AF100756 SEQ ID-Nr. 40 coat protein, gamma-cop 41.EEVKLIKKM 137-145 M11147 SEQ ID-Nr. 41 ferritin, light polypeptide 42.DEVKLPAKL 158-166 AF312393 SEQ ID-Nr. 42 polymerase I and transcriptrelease factor 43. TERELKVAY 637-645 AB040951 SEQ ID-Nr. 43 hypotheticalprotein FLJ20004 44. NEFSLKGVDF 86-95 J04101 SEQ ID-Nr. 44 ets-1 45.NEQDLGIQY 169-177 D13866 SEQ ID-Nr. 45 catenin alpha 1 46. EERIVELF306-313 BC000627 SEQ ID-Nr. 46 signal transducer and activator oftranscription 3 47. EEIREAFRVF 84-93 J04046 SEQ ID-Nr. 47 calmodulin 348. DEYIYRHFF 344-352 AF011794 SEQ ID-Nr. 48 cell cycle progression 8protein 49. DELELHQRF 308-316 X86098 SEQ ID-Nr. 49 adenovirus 5 E1Abinding protein 50. SEVKFTVTF 80-88 M87842 SEQ ID-Nr. 50 galectin 2 51.IETIINTF 12-19 M26311 SEQ ID-Nr. 51 calgranulin B 52. KENPLQFKF61-69/72-80 J05021/ SEQ ID-Nr. 52 villin 2 (ezrin)/(radixin) L02320 53.DEVRTLTY 41-48 Y10807 SEQ ID-Nr. 53 hnRNP methyltransferase, S.cerevisiae-like 2 54. GEAVVNRVF 43-51 Z14977 SEQ ID-Nr. 54 largemultifunctional protease 2, LMP2 55. EEVLIPDQKY 385-394 AF126028 SEQID-Nr. 55 F-box and leucine-rich repeat protein 3A 56. DEGRLVLEF 163-171L21934 SEQ ID-Nr. 56 sterol O-acyltransferase 1 57. DEVELIHF 838-845AF152961 SEQ ID-Nr. 57 chromatin-specific transcription elongationfactor 58. VEVLLNYAY 83-91 AF205218 SEQ ID-Nr. 58 NS 1-binding protein59. TENDIRVMF 120-128 AF267534 SEQ ID-Nr. 59 CUG triplet repeat,RNA-binding protein 1 60. LEGLTVVY 62-69 AF151878 SEQ ID-Nr. 60 coatomerprotein complex subunit zeta 1 61. NELPTVAF 192-199 AK001475 SEQ ID-Nr.61 hypothetical protein 62. EEFGQAFSF 77-85 X03100 SEQ ID-Nr. 62 MHC,class II, DP alpha 1 63. VEAIFSKY 33-40 M29063 SEQ ID-Nr. 63 hnRNP C(C1/C2) 64. DERTFHIFY 277-285 M69181 SEQ ID-Nr. 64 myosin, heavypolypeptide 10, non-muscle 65. TEKVLAAVY 206-214 K01177 SEQ ID-Nr. 65aldolase B, fructose-bisphosphate 66. VESPLSVSF 159-167 AK025971 SEQID-Nr. 66 hypothetical protein FLJ22318 67. SEAGSHTLQW MHC-I SEQ ID-Nr.67 68. DEGKVIRF 56-63 BF431469 SEQ ID-Nr. 68 EST reading frame-1 PatientRCC13 HLA-A*02 69. ALAAVVTEV frameshift, DDX3 reading frame +2 AF061337SEQ ID-Nr. 69 70. TLIEDILGV 209-217 AL132825 SEQ ID-Nr. 70 transientreceptor protein 4 associated protein 71. ALFGALFLA 2-10 L26232 SEQID-Nr. 71 phospholipid transfer protein 72. VLATLVLLL 72-80 AA483794 SEQID-Nr. 72 EST 73. TLDDLIAAV 325-333 AK000904 SEQ ID-Nr. 73 hypotheticalprotein FLJ10042 74. YLDNGVVFV 316-324 U18299 SEQ ID-Nr. 74damage-specific DNA binding protein 1 (127kD) 75. SVFAGVVGV 581-589U58855 SEQ ID-Nr. 75 guanylate cyclase 1, soluble, alpha 3 76.SLINVGLISV 48-57 BC000476 SEQ ID-Nr. 76 acidic protein rich in leucines77. ALADGVQKV 176-184 AF323540 SEQ ID-Nr. 77 apolipoprotein L, 1)HLA-A*24 78. TYGEIFEKE 107-115 AF070652 SEQ ID-Nr. 78 NADH dehydrogenase(ubiquinone) 1, (B14.5b) 79. YYMIGEQKF 203-211 U08021 SEQ ID-Nr. 79nicotinamide-n-methyltransferase

1. Tumor-associated peptide containing an amino acid sequence which isselected from the group consisting of SEQ ID NO:1 to SEQ ID NO:79 of theenclosed sequence listing, the peptide having the ability to bind to amolecule of human major histocompatibility complex (MHC) class-I. 2.Peptide according to claim 1, wherein at least one amino acid isreplaced by another amino acid having similar chemical features. 3.Peptide according to claim 1, wherein said peptide comprises at leastone additional amino acid at the N— or C-terminus.
 4. Peptide accordingto claim 2, wherein said peptide comprises at least one additional aminoacid at the N— or C-terminus.
 5. Peptide according to claim 1, whereinat least one amino acid is deleted.
 6. Peptide according to claim 2,wherein at least one amino acid is deleted.
 7. Peptide according toclaim 3, wherein at least one amino acid is deleted.
 8. Peptideaccording to claim 4, wherein at least one amino acid is deleted. 9.Peptide according to claim 1, wherein at least one amino acid ischemically modified.
 10. Peptide according to claim 2, wherein at leastone amino acid is chemically modified.
 11. Peptide according to claim 3,wherein at least one amino acid is chemically modified.
 12. Peptideaccording to claim 4, wherein at least one amino acid is chemicallymodified.
 13. Peptide according to claim 5, wherein at least one aminoacid is chemically modified.
 14. Peptide according to claim 6, whereinat least one amino acid is chemically modified.
 15. Peptide according toclaim 7, wherein at least one amino acid is chemically modified. 16.Peptide according to claim 8, wherein at least one amino acid ischemically modified.
 17. A method of manufacturing a medicament fortreatment of tumor diseases and/or adenomatous diseases, the methodcomprising the step of providing at least one peptide having an aminoacid sequence which is selected from the group consisting of SEQ ID NO:1to SEQ ID NO:79 in a therapeutically effective amount.
 18. A method fortreating tumor diseases and/or adenomatous diseases, the methodcomprising administering a peptide according to claim
 1. 19. A methodfor treating tumor diseases and/or adenomatous diseases, the methodcomprising administering a peptide according to claim
 2. 20. A methodfor treating tumor diseases and/or adenomatous diseases, the methodcomprising administering a peptide according to claim
 3. 21. A methodfor treating tumor diseases and/or adenomatous diseases, the methodcomprising administering a peptide according to claim
 5. 22. A methodfor treating tumor diseases and/or adenomatous diseases, the methodcomprising administering a peptide according to claim
 9. 23. The methodof claim 17, wherein said disease is selected from the group comprisingof renal, breast, pancreas, gastric, bladder and/or testis cancer. 24.The method of claim 18, wherein said disease is selected from the groupconsisting of renal, breast, pancreas, gastric, bladder and/or testiscancer.
 25. The method of claim 18, wherein said peptide is used incombination with an adjuvant.
 26. The method of claim 23, wherein saidpeptide is used in combination with an adjuvant.
 27. The method of claim18, wherein said peptide is used when bound to an antigen-presentingcell.
 28. The method of claim 23, wherein said peptide is used whenbound to an antigen-presenting cell.
 29. A method for labelling ofleukocytes, in particular T lymphocytes, wherein a peptide according toclaim 1 is used.
 30. The method of claim 29, wherein a therapy course ofa tumor disease is assessed.
 31. A method for preparing an antibody,wherein a peptide according to claim 1 is used.
 32. Pharmaceuticalcomposition comprising one or more peptides according to claim
 1. 33.Pharmaceutical composition comprising one or more peptides according toclaim
 2. 34. Pharmaceutical composition comprising one or more peptidesaccording to claim
 3. 35. Pharmaceutical composition comprising one ormore peptides according to claim
 5. 36. Pharmaceutical compositioncomprising one or more peptides according to claim
 9. 37. Nucleic acidmolecule encoding a peptide according to claim
 1. 38. Pharmaceuticalcomposition comprising nucleic acid molecules according to claim
 37. 39.Vector comprising the nucleic acid molecule according to claim
 37. 40.Cell, genetically modified by means of the nucleic acid moleculeaccording to claim 37 to produce a peptide containing an amino acidsequence which is selected from the group consisting of SEQ ID NO:1 toSEQ ID NO:79 of the enclosed sequence listing.
 41. Cell, geneticallymodified by means of the vector according to claim 39, to produce apeptide containing an amino acid sequence which is selected from thegroup consisting of SEQ ID NO:1 to SEQ ID NO:79 of the enclosed sequencelisting.
 42. A method for killing target cells in a patient, the targetcells aberrantly expressing a polypeptide comprising an amino acidsequence which is selected from the group consisting of SEQ ID NO:1 toSEQ ID NO:79 of the enclosed sequence listing, the method comprising thestep of administering to the patient an effective amount of a peptideaccording to claim 1, wherein the amount of said peptide is effective toinduce an anti-target cell immune response in that patient.
 43. A methodfor killing target cells in a patient, the target cells aberrantlyexpressing a polypeptide comprising an amino acid sequence which isselected from the group consisting of SEQ ID NO:1 to SEQ ID NO:79 of theenclosed sequence listing, the method comprising the step ofadministering to the patient an effective amount of a nucleic acidmolecule according to claim 37, wherein the amount of said nucleic acidmolecule is effective to induce an anti-target cell immune response inthat patient.
 44. An in vitro method for producing activated cytotoxic Tlymphocytes (CTL), the method comprising the step of contacting CTL withantigen loaded MHC class I molecules expressed on the surface of asuitable antigen-presenting cell for a period of time sufficient toactivate, in an antigen specific manner, said CTL wherein said antigenis a peptide according to claim
 1. 45. Activated cytotoxic T lymphocytes(CTL) produced by the method according to claim 44, which selectivelyrecognize a cell which aberrantly express a polypeptide comprising anamino acid sequence which is selected from the group consisting of SEQID NO:1 to SEQ ID NO:79 of the enclosed sequence listing.